1. Field of the Invention
The present invention relates generally to purified protein A compositions and methods for the production of such compositions. In particular, the present invention relates to methods for removing enterotoxins and other contaminants from affinity-purified protein A compositions.
Protein A is a cell surface protein which can be isolated from particular strains of Staphylococcus aureus and which is able to bind free IgG and IgG-complexes. IgG-complexes are antigen-IgG complexes which circulate in patient serum and are not removed by the normal phagocytic mechanisms of the immune system. Protein A is typically obtained by extraction from the cell wall of suitable Staphylococcus aureus strains, or preferably by isolation from the conditioned media of certain Staphylococcus aureus strains which secrete protein A. In either case, the protein A is normally affinity-purified to separate it from other proteins and contaminants. For example, the initial protein A preparation will be passaged over an affinity column having bound human IgG, which specifically binds to the protein A. The column is washed, and the bound protein A then eluted.
Protein A has found wide use in the selective purification of IgG from various biological samples. Additionally, protein A columns have recently been employed for the therapeutic removal of IgG and IgG-containing immune complexes in the treatment of certain cancers and autoimmune diseases. Such treatment protocols call for the removal of a volume of patient blood, separation of the blood into its plasma and cellular components, and perfusion of the plasma over a protein A column for removal of IgG and immune complexes. The cellular and treated plasma components are then reinfused into the patient, with the intention of stimulating the patient's immunne response.
While such protein A plasma perfusion therapies have enjoyed success, they have also been observed to induce certain toxic side effects in the treated patients, such as nausea, vomiting, fevers, chills, and the like. It is suspected that such toxic side effects may result from the presence of contaminating substances in the protein A compositions, particularly staphylococcal enterotoxins, which may leach from the adsorbant columns into the patient plasma during therapy.
The use of protein A as a direct immune system stimulant has also been investigated by injection of protein A into tumor-bearing animals. Although anti-tumor effects have been observed, significant toxic reactions have also been reported. Such toxic reactions appear to result from the presence of enterotoxins and other contaminants in the protein A.
It would therefore be desirable to provide purified protein A compositions and methods for preparing such compositions. In particular, it would be desirable to provide for protein A compositions which are substantially free from staphylococcal enterotoxins and other proteinaceous contaminants.
2. Description of the Background Art
Various reports have indicated that staphylococcal protein A preparations may be contaminated by trace amounts of staphylococcal enterotoxin(s) (Smith et al. (1983) J. Immunol. 130:773; Carlsson (1984) Cell. Immunol. 86:136; Schrezenmeier (1987) J. Immunol. Methods. 105:133). It has recently been suggested that the interferon-inducing capability of protein A derives from the presence of enterotoxin (Carlsson et al., supra.). Moreover, since enterotoxins are extremely potent mitogens, it has been proposed that the mitogenic effects observed with protein A may in fact be due to contamination by enterotoxins (Schrezenmeier et al., supra.). Ion exchange chromatography is a known technique for separating proteins. See, e.g., Hudson and Hay, Practical Immunology, 2nd. ed., Blackwell Scientific Publications, Oxford, 1980, pp. 169-175.